EFFECTS OF GENETIC POLYMORPHISMS ON RNA GUANINE QUADRUPLEX AFFECTING THE TRANSLATION HUMAN ONCOGENS

BEZZI, G.; ARMAS, P.; PIGA, E.J.

online

Congreso; LVI SAIB Meeting - XV SAMIGE Meeting - SAIB-SAMIGE Joint Meeting 2020 on line; 2020

SAIB-SAMIGE

G-quadruplex (G4) nucleic acids are four-stranded secondary structures formed in guanine-rich sequences and prevalent in regulatory regions. G4s have been described as non-canonical RNA secondary structures relevant for translational regulation if present in 5' untranslated regions (5? UTRs) or other mRNA regions involved in translational control and mainly described in oncogenes. On the other hand, genomic scale association studies by massive DNA sequencing revealed that single nucleotide polymorphisms (SPNs) associated with human diseases may be present in 5' UTRs. However, mutations in noncoding regions associated with cancer have been less investigated than those present in coding regions. The goal of this work was to identify SNPs overlapped with putative G4 forming sequences (PG4) described as translational regulators (located within 5' UTRs), that may affect G4 folding, hereafter called SNP-PG4. First we performed a bioinformatics analysis using Ensembl database to identify the SNPs (from COSMIC, ClinVar, dbSNP and HGMD genetic variation databases) overlapped with the PG4s (and their +/- 5 bp flanking sequences) described as transcriptional regulators for 14 oncogenes (ADAM10, CCND3, NRAS, BCL2, H2AFY, MMP16, ESR1, AKTIP, CTSB, FGF2, ZIC1, TRF2, CXCL14 and VEGF). For each reference sequence we generated a collection of variable sequences representing each SNP and a mutant sequence with mutations that abolish PG4 (unable to form G4). Then we used several DNA and RNA G4 folding predictors in order to identify those SNP-PG4 that may affect G4 folding or stability. Using RNAfold, we assessed the effect of these mutations on the thermodynamic stability of predicted RNA G4s in the context of full-length 5′ UTRs. From 245 RNA sequences corresponding to the SNP-PG4 of the analyzed oncogenes we chose 15 for further analysis. The selected sequences correspond to CCND3, NRAS, HSAFY, ESR1, FGF2, ZIC1 and TRF2 oncogenes. Through spectroscopic analyses by Circular Dichroism (CD) and Thermal Difference Spectroscopy (TDS) we demonstrated that some SNPs cause quantitative spectral changes. Moreover, CD melting assays performed with these SNPs indicate that they induce G4 stability changes. In agreement, 1D 1H NMR spectroscopy confirmed that SNPs induce quantitative and qualitative changes for the NRAS SNP-PG4s analyzed. Finally, PG4s were cloned into psiCHECK-2 vector and revealed that luciferase reporter activity was altered by SNPs when transfected into HEK293 cells. Results gathered in this work suggest that SNP-PG4s that alter G4 folding may be the cause of differential expression of oncogenes leading to tumor predisposition, establishment, progression or metastasis, indicating that they could act as cancer driver mutations and should be considered as a novel molecular etiology mechanism for the predisposition or establishment of human diseases.